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J Biol Chem. 2009 Feb 20;284(8):5403-13. doi: 10.1074/jbc.M806012200. Epub 2008 Dec 22.

Analyzing the interaction of RseA and RseB, the two negative regulators of the sigmaE envelope stress response, using a combined bioinformatic and experimental strategy.

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1
Department of Microbiology and Immunology, University of California, San Francisco, California 94158, USA.

Abstract

The Escherichia coli envelope stress response is controlled by the alternative sigma factor, sigma(E), and is induced when unfolded outer membrane proteins accumulate in the periplasm. The response is initiated by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB is an important negative regulator of envelope stress response that exerts its negative effects onsigma(E) activity through its binding to RseA. In this study, we analyze the interaction between RseA and RseB. We found that tight binding of RseB to RseA required intact RseB. Using programs that performed global and local sequence alignment of RseB and RseA, we found regions of high similarity and performed alanine substitution mutagenesis to test the hypothesis that these regions were functionally important. This protocol is based on the hypothesis that functionally dependent regions of two proteins co-evolve and therefore are likely to be sequentially conserved. This procedure allowed us to identify both an N-terminal and C-terminal region in RseB important for binding to RseA. We extensively analyzed the C-terminal region, which aligns with a region of RseA coincident with the major RseB binding determinant in RseA. Both allele-specific suppression analysis and cysteine-mediated disulfide bond formation indicated that this C-terminal region of similarity of RseA and RseB identifies a contact site between the two proteins. We suggest a similar protocol can be successfully applied to pairs of non-homologous but functionally linked proteins to find specific regions of the protein sequences that are important for establishing functional linkage.

PMID:
19103591
PMCID:
PMC2643490
DOI:
10.1074/jbc.M806012200
[Indexed for MEDLINE]
Free PMC Article
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